This year we have continued work on a line of transgenic mice designed to permit the inducible expression of transgenes in the melanocyte stem cell compartment of the hair follicle. (NOTE: Last year, progress on this project was described under the project "Determinants of Melanocyte Transformation and Melanoma Progression and Signal Transduction". It has now evolved into a separate project.) This line of transgenic mice expresses the tetracycline-inducible transactivator, tTA (otherwise known as "Tet-Off"), from the dopachrome tautomerase (Dct) promoter. Since Dct is expressed in the putative melanocyte stem cell in the murine hair follicle, it was envisaged that these mice, if generated successfully, would permit the expression of transgenes driven by the tetracycline-transactivator responsive element (TRE) in these cells.This year we have characterized transgene expression from our transgenic lines using TRE-H2BGFP reporter mice, expressing an H2BGFP fusion protein from the TRE. One line derived from our initial series of founder mice appears promising in that expression of GFP is noted in the outer root sheath of adult hair follicles, consistent with the known localization of melanocytes in this tissue. Expression of GFP is noted in significantly fewer cells following the administration of doxycycline to mice for 3-5 weeks, suggesting that this transgenic line is capable of the regulated expression of transgenes in murine melanocytes. GFP expression in hair follicles following long-term doxycycline administration is limited to small numbers of cells noted in the outer root sheath or adjacent to murine hair follicles. Expression in these "label-retaining cells" implies that they have not undergone significant cell division during the period of doxycycline administration, sharing a slow-cycling property characteristic of other types of adult stem cells.We have also begun to culture primary melanoma tumor lysates in cell culture media designed to optimize their survival properties. Establishing these tumor cell cultures will be important for identifying subpopulations expressing markers characteristic of melanocyte stem cells.